A Guanine-quadruplex located on the negative strand of the hepatitis C virus facilitates efficient genomic RNA synthesis
Bibliographic record
Abstract
Abstract Guanine-rich nucleic acids can form a unique secondary structure called Guanine-quadruplexes (G4s). These G4s are conserved across all domains of life and in viruses, where they can play important regulatory roles in the viral lifecycle. In flaviviruses, the (-) strand 3’ untranslated region (UTR) is essential for initiating genomic RNA synthesis. Within the (-) strand 3’ UTR of hepatitis C virus (HCV), a highly conserved G4 is located within stem-loop IIy’ (SLIIy’), spanning nucleotides 110-131—a region that is essential for efficient replication. Using bioinformatics, we demonstrate that nucleotides 110-131 are highly conserved across HCV genotypes 1-7, and this region within SLIIy’ can likely adopt a more energetically favorable G4 conformation rather than the predicted hairpin. From biophysical and cell biology experiments, we show that stabilizing the predicted hairpin structure in SLIIy’ disrupts RNA synthesis, while successive guanine-to-adenine mutations within the G4 sequence impair G4 formation and hinder replication. Combining our findings with prior studies, we propose that the SLIIy’ G4 plays a crucial role in recruiting NS3 helicase, which is necessary for unwinding RNA structures and facilitating access for NS5B polymerase. The G4 may serve as a regulatory switch that modulates helicase binding and replication efficiency. We propose that in the absence of a stable G4, NS3 recruitment or unwinding is impaired, leading to inefficient RNA unwinding and reduced polymerase activity, ultimately hindering viral replication. These results reveal a previously unrecognized role for G4 structures in the HCV replication cycle, highlighting their importance in regulating (+) strand RNA synthesis. Importance Hepatitis C virus is a positive-sense single-stranded RNA virus that relies on an intermediate negative strand to generate new genomic RNA. While the last 157 nucleotides of the negative-strand 3’ untranslated region are known to be essential for replication, the underlying regulatory mechanisms have remained poorly understood. Our study demonstrates that a highly conserved guanine-rich sequence (107-131 nt) located on the negative strand SLIIy’ forms a guanine-quadruplex secondary structure that plays a pivotal role in orchestrating viral RNA synthesis. This G4 not only promotes efficient replication but likely acts as a molecular target to recruit the NS3 helicase, thereby allowing access for the NS5B polymerase. Disruption of the G4 structure and stabilization of the stem-loop severely impairs viral replication. These findings reveal an unrecognized layer of post-transcriptional regulation in the HCV lifecycle and establish G4 RNA structures as a critical element in HCV genome replication.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".