A Genetic Screen Identifies Interferon-α Effector Genes Required to Suppress Hepatitis C Virus Replication

Interferons (IFNs) restrict various kinds of viral infection via induction of hundreds of IFN-stimulated genes (ISGs), while the functions of the majority of ISGs are broadly unclear. Here, we show that a high-IFN-inducible gene, ISG12a (also known as IFI27), exhibits a nonapoptotic antiviral effect on hepatitis C virus (HCV) infection. Viral NS5A protein is targeted specifically by ISG12a, which mediates NS5A degradation via a ubiquitination-dependent proteasomal pathway. K374R mutation in NS5A domain III abrogates ISG12a-induced ubiquitination and degradation of NS5A. S-phase kinase-associated protein 2 (SKP2) is identified as an ubiquitin E3 ligase for NS5A. ISG12a functions as a crucial adaptor that promotes SKP2 to interact with and degrade viral protein. Moreover, the antiviral effect of ISG12a is dependent on the E3 ligase activity of SKP2. These findings uncover an intriguing mechanism by which ISG12a restricts viral infection and provide clues for understanding the actions of innate immunity. IMPORTANCE Upon virus invasion, IFNs induce numerous ISGs to control viral spread, while the functions of the majority of ISGs are broadlyunclear. The present study shows a novel antiviral mechanism of ISGs and elucidated that ISG12a recruits an E3 ligase, SKP2, for ubiquitination and degradation of viral protein and restricts viral infection. These findings provide important insights into exploring the working principles of innate immunity.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

ISG12a Restricts Hepatitis C Virus Infection through the Ubiquitination-Dependent Degradation Pathway

Xue

Yang

Wang

et al. 2016

“…Huh7 or Huh7.5.1 cells were infected with HIV-based HCVpp bearing envelope glycoproteins of JFH1. Establishment of HCV cell culture has been described previously (20,21). Huh7 and Huh7.5.1 cells were infected with infectious HCV JFH1 at a multiplicity of infection (MOI) of 0.2 or with HCV Jc1/Gluc (22) bearing a secreted Gaussia luciferase reporter at an MOI of 1.…”

Section: Methodsmentioning

confidence: 99%

EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway

Zhu

Xiao

Hong

et al. 2015

Self Cite

The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCVinfected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. H epatitis C virus (HCV) poses a threat to public health by infecting approximately 170 million people worldwide and causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 2). Fifty to ninety percent of acute infections become chronic due to failure to mount a productive immune response to clear the virus. The innate immune system is the first line of defense responsible for recognizing viral pathogens and is therefore one of the crucial elements in determining the outcome of HCV infection.It is known that HCV RNA is recognized by the combined actions of protein kinase R (PKR), retinoic acid-inducible gene 1 (RIG-I), and Toll-like receptor 3 (TLR3) after viral entry into host cells (3-5). Pathogen recognition triggers downstream signaling to activate transcription factors, such as interferon (IFN) regulatory factors (IRFs), to induce type I interferon secretion and stimulation of the JAK-STAT signaling pathway, leading to the expression of IFN-stimulated genes (ISGs), which presumptively mediate control of viral replica-

“…Indeed, recent reports suggest a link between glycolysis and innate immunity. Glucokinase regulatory protein (GCKR), which inhibits HK activity, has been shown to be an IFN-␣ effector gene required to suppress HCV replication in Huh7.5 cells (37). Indeed, inhibition of GCKR expression by siRNA rescues HCV replication from IFN-␣-mediated suppression.…”

Section: Figmentioning

confidence: 99%

Activity of Hexokinase Is Increased by Its Interaction with Hepatitis C Virus Protein NS5A

Ramière

Rodriguez

Enache

et al. 2014

The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (V max and K m ) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection. IMPORTANCESubstantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells. C hronic infection with hepatitis C virus (HCV) is associated with abnormalities in glucidolipidic metabolism that have been shown to play important roles in various aspects of the virus life cycle, such as genome replication, viral assembly, and secretion (1, 2). In vitro, HCV replication is efficiently inhibited by cholesterol and fatty acid biosynthetic pathway inhibitors (3), and release of viral particles from infected cells can be inhibited by modulation of the very-low-density lipoprotein (VLDL) assembly pr...