Characterization of Hepatitis C Virus IRES Quasispecies – From the Individual to the Pool

Abstract

Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus from the genus Hepacivirus. The viral genomic +RNA is 9.6 kb long and contains highly structured 5′ and 3′ untranslated regions (UTRs) and codes for a single large polyprotein, which is co- and post-translationally processed by viral and cellular proteases into at least 11 different polypeptides. Most of the 5′ UTR and an initial part of the polyprotein gene are occupied by an internal ribosome entry site (IRES), which mediates cap-independent translation of the viral proteins and allows the virus to overcome cellular antiviral defense based on the overall reduction of the cap-dependent translation initiation. We reconsidered published results concerning a search for possible correlation between patient response to interferon-based antiviral therapy and accumulation of nucleotide changes within the HCV IRES. However, we were unable to identify any such correlation. Rather than searching for individual mutations, we suggest to focus on determination of individual and collective activities of the HCV IRESs found in patient specimens. We developed a combined, fast, and undemanding approach based on high-throughput cloning of the HCV IRES species to a bicistronic plasmid followed by determination of the HCV IRES activity by flow cytometry. This approach can be adjusted for measurement of the individual HCV IRES activity and for estimation of the aggregate ability of the whole HCV population present in the specimen to synthesize viral proteins. To detect nucleotide variations in the individual IRESs, we used denaturing gradient gel electrophoresis (DGGE) analysis that greatly improved identification and classification of HCV IRES variants in the sample. We suggest that determination of the collective activity of the majority of HCV IRES variants present in one patient specimen in a given time represents possible functional relations among variant sequences within the complex population of viral quasispecies better than bare information about their nucleotide sequences. A similar approach might be used for monitoring of sequence variations in quasispecies populations of other RNA viruses in all cases when changes in primary sequence represent changes in measurable and easily quantifiable phenotypes.

Virological surveillance, molecular phylogeny, and evolutionary dynamics of hepatitis C virus subtypes 1a and 4a isolates in patients from Saudi Arabia

“…Although, RNA secondary and tertiary structure stability is regarded as a significant factor for virus genome stability, but not essential to predict virus stabilization and response to PEG-IFNα therapy ( El Awady et al, 2009 , Zekri et al, 2007 ). Our data are consistent with those of Vopálenský et al ( Vopalensky et al, 2018 ) who demonstrated 102 mutations in HCV subtype 1a IRESs genome isolated from non-responders and 53 mutations in sustained responders to PEG-IFNα plus RBV respectively. El-Tahan et al ( El-Tahan et al, 2018 ) identified 35.7% mutations localized in stem-loop IIIb (nucleotides 172–227) of IRES in isolated HCV subtype 4a Egyptian strains.…”

“…The findings of pair-wise comparison and multiple sequence alignment for nucleotide conservation, nucleotide variations, and positional mutations within representative 5′ UTR sequences were also in an agreement with previous studies of Shier et al (2014 ), El-Tahan et al (2018 ), Moratorio et al (2007 ), Vopalensky et al (2018 ), El Awady et al (2009 ), and Zekri et al (2007 ) which demonstrated nucleotides conservation from 92% to 98% within 5′ UTR region of HCV genome. Interestingly, nucleotide differences were observed as low <6% (i.e.…”

IRES-targeting small molecule inhibits enterovirus 71 replication via allosteric stabilization of a ternary complex

“…Finally, the HCV IRES possess four SL domains. Neither HCV nor FMDV possess a structure similar to EV71 SLII 33,34 . Moreover, the SMs that target HCV appear to function by changing how the IRES binds the 40 S subunit 19 , whereas those that target FMDV increase flexibility of domain 3.…”

Small Molecule Targeting IRES Domain Inhibits Enterovirus 71 Replication via an Allosteric Mechanism that Stabilizes a Ternary Complex

“…By contrast, the HCV IRES is composed of four stem-loops with no structural homology with EV71. HCV does not possess a structure similar to EV71 SLII 32,33 , and thus the mechanism of action of the (DMA-135)-SLII complex is novel relative to small molecules that target the HCV IRES. Moreover, the small molecules that target HCV appear to function by changing how the IRES binds the 40S subunit 19 .…”