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

Abstract

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-alpha 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-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.

Genotype Distribution And 5' Utr Nucleotide Changes In Hepatitis C Virus

“…5' UTR ve IRES bölgesinin mutasyonlarının tedavi yanıtını etkilemesi konusunda literatürde çelişkili sonuçlar mevcuttur. Bazı çalışmalarda 5'UTR bölgesinin mutasyonlarının tedavi cevabını olumlu etkilediği belirtilirken (20,21), bazılarında ise bu bölgede görülen mutasyonlarla tedavi arasın-da bir ilişki saptanmamıştır (22,23).…”

IRES complexity before IFN‐alpha treatment and evolution of the viral load at the early stage of treatment in peripheral blood mononuclear cells from chronic hepatitis C patients

“…Amongst them, it is known that the high rate of virus mutations leads to the generation of a mixture of closely related genomes, the quasispecies, which persist and evolved in the infected patients (Pawlotsky., 2003a;Pawlotsky., 2003c;Rumin et al, 1999;Soler et al, 2002;von Wagner et al, 2003;Vuillermoz et al, 2004). Similar to coding regions, the 5'Non Coding Region (5'NCR) is distributed as a quasispecies. Previous studies have shown that this part of HCV genome might accumulate nucleotide substitutions, (Vizmanos et al, 1998) but it appears to be subjected strong conservatory constraints than the other regions of the HCV genome.…”

“…This is probably due to the need for structural conservation of the mediated translational element: the Internal Ribosome Entry Site (IRES) located in the 5'NC region. This segment (nt 40-372), highly conserved between 3 isolates (Soler et al, 2002), has the potential to form a stable secondary and tertiary structure.…”

Flow cytometric assay using two fluorescent proteins for the function of the internal ribosome entry site of hepatitis C virus

“…Unlike the translation of eukaryotic mRNA starting from the 5 0 -cap structure, the translation of HCV RNA initiates at the internal ribosome entry site (IRES), which encompasses most of the 5 0 -untranslated region (5 0 -UTR) and about 40 nt of the core protein-coding region (3,4). The HCV IRES is highly conserved among HCV isolates in both the primary sequence and secondary structure (5)(6)(7). Since the translation is an essential step in the viral replication and possibly in the pathogenic functions, it would be important to evaluate the activity of HCV IRES for such studies.…”