Key Points
- To determine how domain arrangement within the Hepatitis C Virus (HCV) 5' untranslated region regulates translation initiation and coordinates interactions with host translation factors.
- Constructed domain-swapped mutant variants altering the relative positions of domains II and III within the HCV 5' untranslated region.
- Assessed translation efficiency of the engineered mutants using dicistronic mRNA expression systems.
- Identified and mapped direct RNA-protein binding contacts using ultraviolet crosslinking assays.
- Repositioning domains II and III severely disrupted internal ribosome entry site (IRES) activity, demonstrating that relative domain geometry is required for cap-independent translation.
- Ultraviolet crosslinking demonstrated that domain III directly binds cellular proteins p120 at the apical loop and p170 along the stem portion, independent of domain position or structural context.
- Protein characterization confirmed that p170 and p120 represent subunits of the eukaryotic translation initiation factor eIF3 (p170 and p116/p110).
Structured PICO
PPopulationHepatitis C Virus (HCV) 5' untranslated region (UTR) and eukaryotic translation initiation factor eIF3 models
IInterventionSwapping the position of the two major domains (II and III) on the 5'UTR sequence
CComparatorWild-type 5'UTR sequence
OOutcomeInternal ribosome entry site (IRES) functioning and identification of cellular factors implicated in IRES activitysurrogate
The positioning of domains II and III on the HCV 5'UTR is essential for IRES functioning, and domain III directly interacts with eIF3 subunits p170 and p120.