DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture

Abstract

All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo.Remarkably, HCV replication is not sensitive to IFN-␥ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-␥ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA. IMPORTANCEInterferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

Links between recognition and degradation of cytoplasmic viral RNA in innate immune response

“…These observations indicate that DDX60 promotes both viral RNA degradation and recognition, similar to ZAPS (Figure C). Knockout studies revealed that mouse DDX60 is required for full RIG‐I activation in mouse embryonic fibroblasts and peritoneal macrophages but not bone marrow‐derived dendritic cells , while DDX60 ‐independent RIG‐I activation in mouse cells has also been reported . These findings indicate that DDX60 ‐mediated RIG‐I activation is cell type‐specific.…”

Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases

“…DDX60L neighbors DDX60 on chromosome IV, and its amino acid sequence is B70% homologous to DDX60 [71]. However, our knowledge on functions of DDX60L in innate immunity is still scarce.…”

“…However, our knowledge on functions of DDX60L in innate immunity is still scarce. A study from 2015 describes DDX60L as an innate immune effector of type I, type II, and type III IFN response that acts independently of DDX60 against hepatitis C virus (HCV) in liver cells [71]. The expression of DDX60L is induced soon after the acute HCV infection, during which it represses the viral RNA replication in a manner independent of IRES-mediated translation or RNA stability, and enhances RIG-I-dependent IRF3 activation [71].…”

“…A study from 2015 describes DDX60L as an innate immune effector of type I, type II, and type III IFN response that acts independently of DDX60 against hepatitis C virus (HCV) in liver cells [71]. The expression of DDX60L is induced soon after the acute HCV infection, during which it represses the viral RNA replication in a manner independent of IRES-mediated translation or RNA stability, and enhances RIG-I-dependent IRF3 activation [71]. DDX60L is likely to be highly specific in its antiviral functions, as it was unable to inhibit replication of hepatitis A virus, regardless of its similarity to HCV [71].…”

“…The expression of DDX60L is induced soon after the acute HCV infection, during which it represses the viral RNA replication in a manner independent of IRES-mediated translation or RNA stability, and enhances RIG-I-dependent IRF3 activation [71]. DDX60L is likely to be highly specific in its antiviral functions, as it was unable to inhibit replication of hepatitis A virus, regardless of its similarity to HCV [71]. In addition, it is noteworthy to mention that DDX60L is also upregulated in IFN-β-treated EpsteinBarr-transformed B cells derived from multiple sclerosis patients [174].…”