Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease

Persistent infections with hepatitis C virus (HCV) are likely to depend on viral inhibition of host defenses. We show that the HCV NS3/4A serine protease blocks the phosphorylation and effector action of interferon regulatory factor–3 (IRF-3), a key cellular antiviral signaling molecule. Disruption of NS3/4A protease function by mutation or a ketoamide peptidomimetic inhibitor relieved this blockade and restored IRF-3 phosphorylation after cellular challenge with an unrelated virus. Furthermore, dominant-negative or constitutively active IRF-3 mutants, respectively, enhanced or suppressed HCV RNA replication in hepatoma cells. Thus, the NS3/4A protease represents a dual therapeutic target, the inhibition of which may both block viral replication and restore IRF-3 control of HCV infection

Fanconi anemia proteins function in mitophagy and immunity

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes

Viral and Host Genetic Determinants of Hepatitis C Virus Persistence and Interferon Resistance

Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is an important cause of cirrhosis and hepatocellular carcinoma. HCV replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. The development of the HCV RNA replicon system has allowed the study of persistent HCV RNA replication in tissue culture. We evaluated HCV/IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV genotype 1b subgenomic RNA replicon that differed only at two sites within the NS5A coding region. A replicon with a lysine (K) insertion at HCV codon 2040 (K2040) replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an leucine (L) to serine (S) point mutation at HCV codon 2198 (L2198S) replicated poorly and triggered a cellular response characterized by IFN-! production and low-level interferon-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high passage (HP) variant with 6 additional point mutations throughout the HCV protein-coding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-" treatment and was associated with viral-directed reduction in host cell expression and action of ISG56, an antagonist of HCV RNA translation. We also demonstrated that HCV subgenomic RNA replicons can be used to model the early events of HCV infection. We found that HCV RNA replicons rapidly induce the cellular antiviral response upon their transfection into host Huh7 cells and we determined that intracellular HCV double stranded RNA (dsRNA) is a potent agonist of host dsRNAactivated pathways. A Huh7 derived cell line that is highly permissive for transduction by HCV replicons is specifically defective in the activation of interferon regulatory factor (IRF)-3 by virus infection or HCV dsRNA transfection. We found that a mutation in the caspase recruitment domain (CARD) of the DExH/D-box helicase protein RIG-I, a component of the TLR3-independent intracellular dsRNA-responsive IRF-3 activation pathway, was responsible for this phenotype. Restoration of RIG-I-mediated IRF-3 activation through genetic complementation resulted in decreased permissiveness to HCV RNA replication. These results establish the RIG-I!IRF-3 pathway as a critical determinant of HCV persistence

Alpha Interferon Induces Distinct Translational Control Programs To Suppress Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2α) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2α phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process

Fanconi anemia proteins function in mitophagy and immunity

Fanconi anemia (FA) pathway genes are important tumor suppressors whose best-characterized function is repair of damaged nuclear DNA. Here, we describe an essential role for FA genes in two forms of selective autophagy. Genetic deletion of Fancc blocks the autophagic clearance of viruses (virophagy) and increases susceptibility to lethal viral encephalitis. Fanconi anemia complementation group C (FANCC) protein interacts with Parkin, is required in vitro and in vivo for clearance of damaged mitochondria, and decreases mitochondrial reactive oxygen species (ROS) production and inflammasome activation. The mitophagy function of FANCC is genetically distinct from its role in genomic DNA damage repair. Moreover, additional genes in the FA pathway, including FANCA, FANCF, FANCL, FANCD2, BRCA1, and BRCA2, are required for mitophagy. Thus, members of the FA pathway represent a previously undescribed class of selective autophagy genes that function in immunity and organellar homeostasis. These findings have implications for understanding the pathogenesis of FA and cancers associated with mutations in FA genes

Selective autophagy and viruses

In recent years, the process of selective autophagy has received much attention with respect to the clearance of protein aggregates, damaged mitochondria and bacteria. However, until recently, there have been virtually no studies on the selective autophagy of viruses, although they are perhaps one of the most ubiquitous unwanted constituents in human cells. Recently, we have shown that the ability of neuronal Atg5 to protect against lethal Sindbis virus central nervous system (CNS) infection in mice is associated with impaired viral capsid clearance, increased p62 accumulation and increased neuronal cell death. In vitro, we showed that p62 interacts with the Sindbis capsid protein and targets it for degradation in autophagosomes. Herein, we review these findings and broadly speculate about potential roles of selective viral autophagy in the regulation of host immunity and viral pathogenesis