Temporal regulation of MDA5 inactivation by Caspase-3 dependent cleavage of 14-3-3η.

The kinetics of type I interferon (IFN) induction versus the virus replication compete, and the result of the competition determines the outcome of the infection. Chaperone proteins that involved in promoting the activation kinetics of PRRs rapidly trigger antiviral innate immunity. We have previously shown that prior to the interaction with MAVS to induce type I IFN, 14-3-3η facilitates the oligomerization and intracellular redistribution of activated MDA5. Here we report that the cleavage of 14-3-3η upon MDA5 activation, and we identified Caspase-3 activated by MDA5-dependent signaling was essential to produce sub-14-3-3η lacking the C-terminal helix (αI) and tail. The cleaved form of 14-3-3η (sub-14-3-3η) could strongly interact with MDA5 but could not support MDA5-dependent type I IFN induction, indicating the opposite functions between the full-length 14-3-3η and sub-14-3-3η. During human coronavirus or enterovirus infections, the accumulation of sub-14-3-3η was observed along with the activation of Caspase-3, suggesting that RNA viruses may antagonize 14-3-3η by promoting the formation of sub-14-3-3η to impair antiviral innate immunity. In conclusion, sub-14-3-3η, which could not promote MDA5 activation, may serve as a negative feedback to return to homeostasis to prevent excessive type I IFN production and unnecessary inflammation.

Role of the Chaperone Protein 14-3-3ε in the Regulation of Influenza A Virus-Activated Beta Interferon.

The NS1 protein of the influenza A virus plays a critical role in regulating several biological processes in cells, including the type I interferon (IFN) response. We previously profiled the cellular factors that interact with the NS1 protein of influenza A virus and found that the NS1 protein interacts with proteins involved in RNA splicing/processing, cell cycle regulation, and protein targeting processes, including 14-3-3ε. Since 14-3-3ε plays an important role in retinoic acid-inducible gene I (RIG-I) translocation to mitochondrial antiviral-signaling protein (MAVS) to activate type I IFN expression, the interaction of the NS1 and 14-3-3ε proteins may prevent the RIG-I-mediated IFN response. In this study, we confirmed that the 14-3-3ε protein interacts with the N-terminal domain of the NS1 protein and that the NS1 protein inhibits RIG-I-mediated IFN-ß promoter activation in 14-3-3ε-overexpressing cells. In addition, our results showed that knocking down 14-3-3ε can reduce IFN-ß expression elicited by influenza A virus and enhance viral replication. Furthermore, we found that threonine in the 49th amino acid position of the NS1 protein plays a role in the interaction with 14-3-3ε. Influenza A virus expressing C terminus-truncated NS1 with a T49A mutation dramatically increases IFN-ß mRNA in infected cells and causes slower replication than that of virus without the T-to-A mutation. Collectively, this study demonstrates that 14-3-3ε is involved in influenza A virus-initiated IFN-ß expression and that the interaction of the NS1 protein and 14-3-3ε may be one of the mechanisms for inhibiting type I IFN activation during influenza A virus infection. IMPORTANCE Influenza A virus is an important human pathogen causing severe respiratory disease. The virus has evolved several strategies to dysregulate the innate immune response and facilitate its replication. We demonstrate that the NS1 protein of influenza A virus interacts with the cellular chaperone protein 14-3-3ε, which plays a critical role in retinoic acid-inducible gene I (RIG-I) translocation that induces type I interferon (IFN) expression, and that NS1 protein prevents RIG-I translocation to the mitochondrial membrane. The interaction site for 14-3-3ε is the RNA-binding domain (RBD) of the NS1 protein. Therefore, this research elucidates a novel mechanism by which the NS1 RBD mediates IFN-ß suppression to facilitate influenza A viral replication. Additionally, the findings reveal the antiviral role of 14-3-3ε during influenza A virus infection.

Intracellular innate immunity and mechanism of action of cytosolic nucleic acid receptor-mediated type I IFN against viruses.

The induction of type I interferons (IFN) is critical for antiviral innate immune response. The rapid activation of antiviral innate immune responses is the key to successful clearance of evading pathogens. To achieve this, a series of proteins, including the pathogen recognition receptors (PRRs), the adaptor proteins, the accessory proteins, kinases, and the transcription factors, are all involved and finely orchestrated. The magnitude and latitude of type I IFN induction however are distinctly regulated in different tissues. A set of interferon simulated genes (ISGs) are then expressed in response to type I IFN signaling to set the cells in the antiviral state. In this review, how type I IFN is induced by viral infections by intracellular PRRs and how type I IFN triggers the expression of downstream effectors will be discussed.

Genotypic Regulation of Type I Interferon Induction Pathways by Frameshift (F) Proteins of Hepatitis C Virus.

Hepatitis C virus (HCV) has evolved mechanisms to evade innate immunity that are leading to chronic infections. The immunological function of the HCV frameshift (F) protein, which is a frameshift product of core coding sequences, has not been well characterized. The HCV F protein is produced during natural HCV infections and is found most commonly in genotype 1 HCV. In this study, we investigated whether the F protein plays a role in type I interferon (IFN) induction pathways. We engineered F expression constructs from core coding sequences of 4 genotypes (1a, 2a, 3a, and 4a) of HCV as well as the sequences which would only be able to produce core proteins. The peptide lengths and amino acids sequences of F proteins are highly variable. We hypothesized that F proteins from different genotypes might control the type I IFN production and response differently. We found that both IFN-beta (IFN-ß) promoter activities are significantly higher in genotype 1a F protein (F1a)-expressing cells. Conversely, the IFN-ß promoter activities are lower in genotype 2a F (F2a) protein-expressing cells. We also used real-time PCR to confirm IFN-ß mRNA expression levels. By generating chimera F proteins, we discovered that the effects of F proteins were determined by the amino acid sequence 40 to 57 of genotype 1a. The regulation of type I IFN induction pathway is related but not limited to the activity of F1a to interact with proteasome subunits and to disturb the proteasome activity. Further molecular mechanisms of how F proteins from different genotypes of HCV control these pathways differently remain to be investigated.IMPORTANCE Although naturally present in HCV infection patient serum, the virological or immunological functions of the HCV F protein, which is a frameshift product of core coding sequences, remain unclear. Here, we report the effects of the HCV F protein between genotypes and discuss a potential explanation for the differential responses to type I IFN-based therapy among patients infected with different genotypes of HCV. Our study provides one step forward to understanding the host response during HCV infection and new insights for the prediction of the outcome of IFN-based therapy in HCV patients.

Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Suppresses Type I and Type III Interferon Induction by Targeting RIG-I Signaling.

Type I and type III interferons (IFNs) are the frontline of antiviral defense mechanisms that trigger hundreds of downstream antiviral genes. In this study, we observed that MERS-CoV nucleocapsid (N) protein suppresses type I and type III IFN gene expression. The N protein suppresses Sendai virus-induced IFN-ß and IFN-λ1 by reducing their promoter activity and mRNA levels, as well as downstream IFN-stimulated genes (ISGs). Retinoic acid-inducible gene I (RIG-I) is known to recognize viral RNA and induce IFN expression through tripartite motif-containing protein 25 (TRIM25)-mediated ubiquitination of RIG-I caspase activation and recruitment domains (CARDs). We discovered that MERS-CoV N protein suppresses RIG-I-CARD-induced, but not MDA5-CARD-induced, IFN-ß and IFN-λ1 promoter activity. By interacting with TRIM25, N protein impedes RIG-I ubiquitination and activation and inhibits the phosphorylation of transcription factors IFN-regulatory factor 3 (IRF3) and NF-κB that are known to be important for IFN gene activation. By employing a recombinant Sindbis virus-EGFP replication system, we showed that viral N protein downregulated the production of not only IFN mRNA but also bioactive IFN proteins. Taken together, MERS-CoV N protein functions as an IFN antagonist. It suppresses RIG-I-induced type I and type III IFN production by interfering with TRIM25-mediated RIG-I ubiquitination. Our study sheds light on the pathogenic mechanism of how MERS-CoV causes disease.IMPORTANCE MERS-CoV causes death of about 35% of patients. Published studies showed that some coronaviruses are capable of suppressing interferon (IFN) expression in the early phase of infection and MERS-CoV proteins can modulate host immune response. In this study, we demonstrated that MERS-CoV nucleocapsid (N) protein suppresses the production of both type I and type III IFNs via sequestering TRIM25, an E3 ubiquitin ligase that is essential for activating the RIG-I signaling pathway. Ectopic expression of TRIM25 rescues the suppressive effect of the N protein. In addition, the C-terminal domain of the viral N protein plays a pivotal role in the suppression of IFN-ß promoter activity. Our findings reveal how MERS-CoV evades innate immunity and provide insights into the interplay between host immune response and viral pathogenicity.

The 14-3-3η chaperone protein promotes antiviral innate immunity via facilitating MDA5 oligomerization and intracellular redistribution.

MDA5 belongs to the RIG-I-like receptor family and plays a non-redundant role in recognizing cytoplasmic viral RNA to induce the production of type I IFNs. Upon RNA ligand stimulation, we observed the redistribution of MDA5 from the cytosol to mitochondrial membrane fractions. However, the molecular mechanisms of MDA5 activation remain less understood. Here we show that 14-3-3η is an essential accessory protein for MDA5-dependent type I IFN induction. We found that several 14-3-3 isoforms may interact with MDA5 through the CARDs (N-MDA5), but 14-3-3η was the only isoform that could enhance MDA5-dependent IFNß promoter activities in a dose-dependent manner. Knock-down of 14-3-3η in Huh7 cells impaired and delayed the kinetics of MDA5 oligomerization, which is a critical step for MDA5 activation. Consequently, the MDA5-dependent IFNß promoter activities as well as IFNß mRNA expression level were also decreased in the 14-3-3η knocked-down cells. We also demonstrated that 14-3-3η is essential in boosting the activation of MDA5-dependent antiviral innate immunity during viral infections. In conclusion, our results uncover a novel function of 14-3-3η to promote the MDA5-dependent IFNß induction pathway by reducing the immunostimulatory potential of viral dsRNA within MDA5 activation signaling pathway.

HCV-induced autophagosomes are generated via homotypic fusion of phagophores that mediate HCV RNA replication.

Hepatitis C virus (HCV) induces autophagy to promote its replication, including its RNA replication, which can take place on double-membrane vesicles known as autophagosomes. However, how HCV induces the biogenesis of autophagosomes and how HCV RNA replication complex may be assembled on autophagosomes were largely unknown. During autophagy, crescent membrane structures known as phagophores first appear in the cytoplasm, which then progress to become autophagosomes. By conducting electron microscopy and in vitro membrane fusion assay, we found that phagophores induced by HCV underwent homotypic fusion to generate autophagosomes in a process dependent on the SNARE protein syntaxin 7 (STX7). Further analyses by live-cell imaging and fluorescence microscopy indicated that HCV-induced phagophores originated from the endoplasmic reticulum (ER). Interestingly, comparing with autophagy induced by nutrient starvation, the progression of phagophores to autophagosomes induced by HCV took significantly longer time, indicating fundamental differences in the biogenesis of autophagosomes induced by these two different stimuli. As the knockdown of STX7 to inhibit the formation of autophagosomes did not affect HCV RNA replication, and purified phagophores could mediate HCV RNA replication, the assembly of the HCV RNA replication complex on autophagosomes apparently took place during the formative stage of phagophores. These findings provided important information for understanding how HCV controlled and modified this important cellular pathway for its own replication.

Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus.

RIG-I is a cytosolic pathogen recognition receptor that engages viral RNA in infected cells to trigger innate immune defenses through its adaptor protein MAVS. MAVS resides on mitochondria and peroxisomes, but how its signaling is coordinated among these organelles has not been defined. Here we show that a major site of MAVS signaling is the mitochondrial-associated membrane (MAM), a distinct membrane compartment that links the endoplasmic reticulum to mitochondria. During RNA virus infection, RIG-I is recruited to the MAM to bind MAVS. Dynamic MAM tethering to mitochondria and peroxisomes then coordinates MAVS localization to form a signaling synapse between membranes. Importantly, the hepatitis C virus NS3/4A protease, which cleaves MAVS to support persistent infection, targets this synapse for MAVS proteolysis from the MAM, but not from mitochondria, to ablate RIG-I signaling of immune defenses. Thus, the MAM mediates an intracellular immune synapse that directs antiviral innate immunity.

Pipette-operable microfluidic devices with hydrophobic valves in sequential dispensing with various liquid samples: multiplex disease assay by RT-LAMP.

Microfluidic dispensing technologies often require additional equipment, posing challenges for their integration into point-of-care testing (POCT) applications. In response to this challenge, we have developed a pipette-operable microfluidic device fabricated using 3D printing technology for precise liquid dispensing. This device features three reaction chambers and three distinct hydrophobic valves to control the flow direction of liquids. Through these valves, the pipette-operable microfluidic device can sequentially dispense and isolate the liquid into the three reaction chambers, allowing for the individual conduction of three distinct reactions. These hydrophobic valves, with optimized flow resistance and burst pressure, can sustain a volumetric flow rate of up to 25 µL s-1, making them compatible with a standard pipette, a syringe, or a dropper operation. Furthermore, the device is successfully used to operate with various liquids, including BSA, DMEM, FBS, plasma, and blood, representing that the device has the potential to be used for various applications. Additionally, distinct RT-LAMP primer sets have been incorporated for diagnosing SARS-CoV-2, influenza A, and influenza B within each chamber through lyophilization. This pipette-operable microfluidic device serves as a versatile tool for diagnosing these three diseases using a single loading process, with results readable by the naked eye or image assay within 30 minutes of incubation. Finally, the design concepts are extended to engineer a microfluidic device with 20 reaction chambers, offering significant potential for multi-disease diagnostics.

Replication of hepatitis C virus RNA on autophagosomal membranes.

Previous studies indicated that hepatitis C virus (HCV) perturbs the autophagic pathway to induce the accumulation of autophagosomes in cells. To understand the role of autophagosomes in the HCV life cycle, we established a stable Huh7 hepatoma cell line that contained an HCV subgenomic RNA replicon and also expressed a GFP-LC3 fusion protein. The GFP-LC3 protein is localized to autophagosomes during autophagy and served as a convenient marker for autophagosomes. Our results indicate that the silencing of the expression of LC3 or Atg7, two protein factors critical for the formation of autophagosomes, suppresses the replication of HCV RNA. Confocal microscopy studies revealed the localization of HCV NS5A and NS5B proteins, which are two important components of the HCV RNA replication complex, and nascent HCV RNA to autophagosomes. The association of the HCV RNA replication complex with the autophagosomal membranes was further confirmed by co-immunoprecipitation and immunoelectron microscopy studies. Interestingly, inhibition of Class III PI3K activity had no effect on the autophagosomes induced by HCV. These results indicate that HCV induces autophagosomes via a Class III PI3K-independent pathway and uses autophagosomal membranes as sites for its RNA replication.

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog.

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.

Interactome Profiling of N-Terminus-Truncated NS1 Protein of Influenza A Virus Reveals Role of 14-3-3γ in Virus Replication.

Influenza A virus is transmitted through a respiratory route and has caused several pandemics throughout history. The NS1 protein of influenza A virus, which consists of an N-terminal RNA-binding domain and a C-terminal effector domain, is considered one of the critical virulence factors during influenza A virus infection because the viral protein can downregulate the antiviral response of the host cell and facilitate viral replication. Our previous study identified an N-terminus-truncated NS1 protein that covers the C-terminus effector domain. To comprehensively explore the role of the truncated NS1 in cells, we conducted immunoprecipitation coupled with LC-MS/MS to identify its interacting cellular proteins. There were 46 cellular proteins identified as the components of the truncated NS1 protein complex. As for our previous results for the identification of the full-length NS1-interacting host proteins, we discovered that the truncated NS1 protein interacts with the γ isoform of the 14-3-3 protein family. In addition, we found that the knockdown of 14-3-3γ in host cells reduced the replication of the influenza A/PR8 wild-type virus but not that of the PR8-NS1/1-98 mutant virus, which lacks most of the effector domain of NS1. This research highlights the role of 14-3-3γ, which interacts with the effector domain of NS1 protein, in influenza A viral replication.

Digital Microfluidic qPCR Cartridge for SARS-CoV-2 Detection.

Point-of-care (POC) tests capable of individual health monitoring, transmission reduction, and contact tracing are especially important in a pandemic such as the coronavirus disease 2019 (COVID-19). We develop a disposable POC cartridge that can be mass produced to detect the SARS-CoV-2 N gene through real-time quantitative polymerase chain reaction (qPCR) based on digital microfluidics (DMF). Several critical parameters are studied and improved, including droplet volume consistency, temperature uniformity, and fluorescence intensity linearity on the designed DMF cartridge. The qPCR results showed high accuracy and efficiency for two primer-probe sets of N1 and N2 target regions of the SARS-CoV-2 N gene on the DMF cartridge. Having multiple droplet tracks for qPCR, the presented DMF cartridge can perform multiple tests and controls at once.

The transmembrane serine protease hepsin suppresses type I interferon induction by cleaving STING.

Many viral proteases mediate the evasion of antiviral innate immunity by cleaving adapter proteins in the interferon (IFN) induction pathway. Host proteases are also involved in innate immunity and inflammation. Here, we report that the transmembrane protease hepsin (also known as TMPRSS1), which is predominantly present in hepatocytes, inhibited the induction of type I IFN during viral infections. Knocking out hepsin in mouse embryonic fibroblasts (MEFs) increased the viral infection-induced expression of Ifnb1, an Ifnb1 promoter reporter, and an IFN-sensitive response element promoter reporter. Ectopic expression of hepsin in cultured human hepatocytes and HEK293T cells suppressed the induction of IFNß during viral infections by reducing the abundance of STING. These effects depended on the protease activity of hepsin. We identified a putative hepsin target site in STING and showed that mutating this site protected STING from hepsin-mediated cleavage. In addition to hepatocytes, several hepsin-producing prostate cancer cell lines showed reduced STING-mediated type I IFN induction and responses. These results reveal a role for hepsin in suppressing STING-mediated type I IFN induction, which may contribute to the vulnerability of hepatocytes to chronic viral infections.

The Molecular Basis of Viral Inhibition of IRF- and STAT-Dependent Immune Responses.

The antiviral innate immunity is the first line of host defense against virus infections. In mammalian cells, viral infections initiate the expression of interferons (IFNs) in the host that in turn activate an antiviral defense program to restrict viral replications by induction of IFN stimulated genes (ISGs), which are largely regulated by the IFN-regulatory factor (IRF) family and signal transducer and activator of transcription (STAT) family transcription factors. The mechanisms of action of IRFs and STATs involve several post-translational modifications, complex formation, and nuclear translocation of these transcription factors. However, many viruses, including human immunodeficiency virus (HIV), Zika virus (ZIKV), and herpes simplex virus (HSV), have evolved strategies to evade host defense, including alteration in IRF and STAT post-translational modifications, disturbing the formation and nuclear translocation of the transcription complexes as well as proteolysis/degradation of IRFs and STATs. In this review, we discuss and summarize the molecular mechanisms by which how viral components may target IRFs and STATs to antagonize the establishment of antiviral host defense. The underlying host-viral interactions determine the outcome of viral infection. Gaining mechanistic insight into these processes will be crucial in understanding how viral replication can be more effectively controlled and in developing approaches to improve virus infection outcomes.

Regulation of Retinoic Acid Inducible Gene-I (RIG-I) Activation by the Histone Deacetylase 6.

Retinoic acid inducible gene-I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the immune response against many RNA viruses. Upon RNA ligand binding, RIG-I undergoes a conformational change facilitating its homo-oligomerization and activation that results in its translocation from the cytosol to intracellular membranes to bind its signaling adaptor protein, mitochondrial antiviral-signaling protein (MAVS). Here we show that RIG-I activation is regulated by reversible acetylation. Acetyl-mimetic mutants of RIG-I do not form virus-induced homo-oligomers, revealing that acetyl-lysine residues of the RIG-I repressor domain prevent assembly to active homo-oligomers. During acute infection, deacetylation of RIG-I promotes its oligomerization upon ligand binding. We identify histone deacetylase 6 (HDAC6) as the deacetylase that promotes RIG-I activation and innate antiviral immunity to recognize and restrict RNA virus infection.

Hepatitis C virus translation preferentially depends on active RNA replication.

Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

The mitochondrial targeting chaperone 14-3-3ε regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.

RIG-I is a cytosolic pathogen recognition receptor that initiates immune responses against RNA viruses. Upon viral RNA recognition, antiviral signaling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I-binding partner and essential component of a translocation complex or "translocon" containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase. The RIG-I translocon directs RIG-I redistribution from the cytosol to membranes where it mediates MAVS-dependent innate immune signaling during acute RNA virus infection. 14-3-3ε is essential for the stable interaction of RIG-I with TRIM25, which facilitates RIG-I ubiquitination and initiation of innate immunity against hepatitis C virus and other pathogenic RNA viruses. Our results define 14-3-3ε as a key component of a RIG-I translocon required for innate antiviral immunity.

Hepatitis C Virus Evasion from RIG-I-Dependent Hepatic Innate Immunity.

Exposure to hepatitis C virus (HCV) usually results in persistent infection that often develops into chronic liver disease. Interferon-alpha (IFN) treatment comprises the foundation of current approved therapy for chronic HCV infection but is limited in overall efficacy. IFN is a major effector of innate antiviral immunity and is naturally produced in response to viral infection when viral pathogen-associated molecular patterns (PAMPs) are recognized as nonself and are bound by cellular pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs) and the RIG-I-like receptors (RLRs). Within hepatocytes, RIG-I is a major PRR of HCV infection wherein PAMP interactions serve to trigger intracellular signaling cascades in the infected hepatocyte to drive IFN production and the expression of interferon-stimulated genes (ISGs). ISGs function to limit virus replication, modulate the immune system, and to suppress virus spread. However, studies of HCV-host interactions have revealed several mechanisms of innate immune regulation and evasion that feature virus control of PRR signaling and regulation of hepatic innate immune programs that may provide a molecular basis for viral persistence.