A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness
Abstract
We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReED's mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that the ReED in NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.
Related
- The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro
- Structural evolution of LEAFY reveals DNA-mediated cooperativity and dimerization shifts at the water-to-land transition
- The proteasome and ERAD pathways can regulate hepatitis E virus pORF1 proteostasis and genome replication
- RNF152 regulates prototype foamy virus replication by targeting Gag polyubiquitination and VLPs production
- Dynamic phosphorylation of the Plasmodium falciparum Myosin A motor regulates motor speed and is important for asexual replication
BibTeX
@article{rothhaar2026novel,
title = {{A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness}},
author = {Paul Rothhaar and Thibault Tubiana and Colin Förster and Gabriel Vanegas Arias and Tomke Arand and Noemi Schäfer and Christian Heuss and Antonio Piras and Andreas Pichlmair and Xavier Hanoulle and Stéphane Bressanelli and Volker Lohmann},
journal = {PLoS Pathogens},
year = {2026},
month = aug,
doi = {10.1371/journal.ppat.1014239},
url = {https://hal.science/hal-05845035v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness
AU - Paul Rothhaar
AU - Thibault Tubiana
AU - Colin Förster
AU - Gabriel Vanegas Arias
AU - Tomke Arand
AU - Noemi Schäfer
AU - Christian Heuss
AU - Antonio Piras
AU - Andreas Pichlmair
AU - Xavier Hanoulle
AU - Stéphane Bressanelli
AU - Volker Lohmann
PY - 2026
DA - 2026/08/05
JO - PLoS Pathogens
DO - 10.1371/journal.ppat.1014239
UR - https://hal.science/hal-05845035v1
AB - We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReED's mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that the ReED in NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.
SN - 1553-7366
SN - 1553-7374
ER -