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Regulation of PKR and IRF-1 during hepatitis C virus RNA replication
Jill Pflugheber1, Brenda Fredericksen, Rhea Sumpter
1Departments of Microbiology and Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9048, USA.
Abstract:
The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.
Insights
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) blocks a key antiviral pathway involving protein kinase R (PKR) and IFN regulatory factor-1 (IRF-1). This disruption allows HCV RNA replication, contributing to viral persistence.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Hepatitis C virus (HCV) replication mechanisms and host interactions are not fully understood.
- Innate antiviral responses are crucial for controlling viral infections but can be subverted by viruses.
- The role of intracellular antiviral signaling pathways in HCV pathogenesis requires further investigation.
Purpose of the Study:
- To investigate cellular antiviral pathways activated during HCV RNA replication.
- To identify viral factors that interfere with host antiviral responses.
- To elucidate the mechanism by which HCV evades innate immunity.
Main Methods:
- Utilized subgenomic HCV replicons in cultured human hepatoma cells.
- Examined double-stranded RNA (dsRNA) signaling pathways and their components, including protein kinase R (PKR) and IFN regulatory factor-1 (IRF-1).
- Investigated the role of the viral nonstructural protein 5A (NS5A) in modulating dsRNA signaling and PKR activation.
Main Results:
- HCV RNA replication activated dsRNA signaling pathways.
- Viral RNA replication efficiency correlated with the ability to suppress dsRNA-induced PKR and IRF-1 activation.
- The viral NS5A protein was identified as the key inhibitor, binding to PKR and blocking dsRNA-mediated activation of IRF-1.
- Mutations in the NS5A PKR-binding domain restored IRF-1 activation and reduced HCV RNA replication.
Conclusions:
- HCV NS5A protein actively disrupts the host's innate antiviral response by inhibiting the PKR/IRF-1 signaling pathway.
- This disruption of host antiviral mechanisms by NS5A likely contributes to HCV persistence.
- Targeting NS5A interactions with host factors could be a therapeutic strategy against HCV.