MicroRNAs (miRNAs) are small noncoding RNAs that regulate eukaryotic gene expression by binding to regions of imperfect complementarity in mRNAs, typically in the 3′ UTR, recruiting an Argonaute (Ago) protein complex that usually results in translational repression or destabilization of the target RNA. The translation and decay of mRNAs are closely linked, competing processes, and whether the miRNA-induced silencing complex (RISC) acts primarily to reduce translation or stability of the mRNA remains controversial. miR-122 is an abundant, liver-specific miRNA that is an unusual host factor for hepatitis C virus (HCV), an important cause of liver disease in humans. Prior studies show that it binds the 5′ UTR of the messenger-sense HCV RNA genome, stimulating translation and promoting genome replication by an unknown mechanism. Here we show that miR-122 binds HCV RNA in association with Ago2 and that this slows decay of the viral genome in infected cells. The stabilizing action of miR-122 does not require the viral RNA to be translationally active nor engaged in replication, and can be functionally substituted by a nonmethylated 5′ cap. Our data demonstrate that a RISC-like complex mediates the stability of HCV RNA and suggest that Ago2 and miR-122 act coordinately to protect the viral genome from 5′ exonuclease activity of the host mRNA decay machinery. miR-122 thus acts in an unconventional fashion to stabilize HCV RNA and slow its decay, expanding the repertoire of mechanisms by which miRNAs modulate gene expression.

Hepatitis C virus (HCV) replication is dependent on microRNA 122 (miR-122), a liver-specific microRNA that recruits Argonaute 2 to the 5′ end of the viral genome, stabilizing it and slowing its decay both in cell-free reactions and in infected cells. Here we describe the RNA degradation pathways against which miR-122 provides protection. Transfected HCV RNA is degraded by both the 5′ exonuclease Xrn1 and 3′ exonuclease exosome complex, whereas replicating RNA within infected cells is degraded primarily by Xrn1 with no contribution from the exosome. Consistent with this, sequencing of the 5′ and 3′ ends of RNA degradation intermediates in infected cells confirmed that 5′ decay is the primary pathway for HCV RNA degradation. Xrn1 knockdown enhances HCV replication, indicating that Xrn1 decay and the viral replicase compete to set RNA abundance within infected cells. Xrn1 knockdown and miR-122 supplementation have equal, redundant, and nonadditive effects on the rate of viral RNA decay, indicating that miR-122 protects HCV RNA from 5′ decay. Nevertheless, Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding, indicating that miR-122 has additional yet uncharacterized function(s) in the viral life cycle.host factor | RNA decay | translation | viral replicase H epatitis C virus (HCV) is a positive-strand RNA virus classified in the family Flaviviridae. It is highly hepatotropic and an important cause of human liver disease (1). Its replication is uniquely dependent on miR-122, which is the most abundant microRNA (miRNA) in the liver and accounts for >50% of mature miRNAs in human hepatocytes (2, 3). There are two conserved miR-122 binding sites (S1 and S2) located near the 5′ end of the positive-sense viral RNA genome, immediately upstream of an internal ribosome entry site (IRES) that mediates translation of the viral polyprotein. Direct interactions between the miR-122 seed sequence (nts 2-8) and these sites in the 5′ UTR are essential for amplification of the HCV genome (4, 5). Additional supplemental base-pairing upstream of S1 and S2 has also been demonstrated and is important for viral replication (6, 7).Unlike typical interactions of miRNAs with mRNAs that involve binding within the 3′ UTR and promote translational repression and/or destabilization of the target RNA (8), binding of miR-122 to the 5′ UTR of HCV genomic RNA stimulates viral protein expression (4, 9) and, in association with Argonaute 2 (Ago2) protein, stabilizes HCV RNA (10). The rate of decay of either transfected synthetic genomic RNA or replicating viral RNA within infected cells is slowed when cells are transfected with synthetic duplex miR-122, thereby supplementing its endogenous abundance. Conversely, transfection of antisense RNA complementary to miR-122 enhances the rate with which HCV RNA decays in either context. HCV RNA is not thought to contain a 5′ cap structure, and the stabilizing action of miR-122 on synthetic RNA can be functionally substituted by addition of a 5′ cap analog (10). These obser...

Background & Aims-Several small molecule inhibitors of the hepatitis C virus (HCV) NS3/4A protease have advanced successfully to clinical trials. However, the selection of drugresistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mutations also adversely affect infectious virus assembly and release, processes in which NS3 also participates.

Hepatitis C virus (HCV) infects over 170 million people worldwide and is a leading cause of liver disease and cancer. The virus has a 9,650-nt, single-stranded, messenger-sense RNA genome that is infectious as an independent entity. The RNA genome has evolved in response to complex selection pressures, including the need to maintain structures that facilitate replication and to avoid clearance by cell-intrinsic immune processes. Here we used highthroughput, single-nucleotide resolution information to generate and functionally test data-driven structural models for three diverse HCV RNA genomes. We identified, de novo, multiple regions of conserved RNA structure, including all previously characterized cisacting regulatory elements and also multiple novel structures required for optimal viral fitness. Well-defined RNA structures in the central regions of HCV genomes appear to facilitate persistent infection by masking the genome from RNase L and double-stranded RNA-induced innate immune sensors. This work shows how structure-first comparative analysis of entire genomes of a pathogenic RNA virus enables comprehensive and concise identification of regulatory elements and emphasizes the extensive interrelationships among RNA genome structure, viral biology, and innate immune responses.RNA structure | evolution | motif discovery | functional validation H epatitis C virus (HCV) currently infects over 170 million people. There is no vaccine, and therapy, generally involving treatment with IFN and ribavirin, is often ineffective (1). Efficacious anti-HCV therapeutics are becoming available (2), but the extent to which they will mitigate the hepatitis C disease burden remains to be seen. Roughly 70% of acutely infected individuals fail to clear the virus and become lifelong HCV carriers, at risk for progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (3).HCV genomes are single-stranded, ∼9,650-nt, messengersense RNA molecules (4). The naked RNA initiates autonomous replication when transfected into cells and establishes chronic HCV infection in chimpanzees (5, 6). The HCV genomic RNA carries genetic information at two levels: a single large ORF encodes viral proteins and complex RNA structural elements regulate the viral replication cycle (4). Viral replication begins when conserved RNA elements in the 5′ UTR bind the 40S ribosome subunit and recruit essential translation factors (7). Translation produces a viral polyprotein that is cleaved by cellular and viral proteases to generate 10 viral proteins (4). The HCV genome is replicated through a negative-strand RNA intermediate by a viral RNA-dependent RNA polymerase (NS5B) in a process controlled by conserved RNA elements (8-17).The HCV genomic RNA is physically compact (18) and highly structured (19). These features likely facilitate persistent HCV infections in humans by protecting the genome from degradation by innate antiviral defenses (20,21). Two elements of this defense are RNase L, which cleaves in single-stranded regions (22), and diverse double...

Although oxidative tissue injury often accompanies viral infection, there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase 2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals, suggesting critical regulation of the conformation of the NS3/4A protease and NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to trans-membrane and membrane-proximal residues within these proteins, and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a novel mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1-2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3-4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10-20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus-host interactions within the liver.innate immunity | viral persistence | immune evasion H epatitis A virus (HAV) is a small, hepatotropic positivestrand RNA virus. Although it is classified among the Picornaviridae, it differs in several important respects from most other human picornaviral pathogens in having a very slow and nonlytic replication cycle (reviewed in ref. 1). Several primatederived cell lines are permissive for infection by HAV, and in the absence of extensive adaptation of the virus to cell culture, these infections are typically noncytopathic. Despite this, HAV causes only acute disease in humans and has never been documented to establish long-term persistent infection within the liver (1-3). However, relatively little is known about the pathogenesis of HAV infections and how the virus is cleared from the liver as there has been little impetus for research on hepatitis A since the development of effective inactivated vaccines almost 2 decades ago.The absence of long-term persistent HAV infection is well documented by disappearance of the virus from isolated human populations over time (4, 5) and differs dramatically from the outcome of hepatitis C virus (HCV) infections, which persist for life in the majority of persons (6). HCV is the cause of considerable human morbidity and mortality. Like HAV, it...

SUMMARY The liver-specific microRNA, miR-122, stabilizes hepatitis C virus (HCV) RNA genomes by recruiting host argonaute 2 (AGO2) to the 5′ end and preventing decay mediated by exonuclease Xrn1. However, HCV replication requires miR-122 in Xrn1-depleted cells, indicating additional function s. We show that miR-122 enhances HCV RNA levels by altering the fraction of HCV genomes available for RNA synthesis. Exogenous miR-122 increases viral RNA and protein levels in Xrn1-depleted cells, with enhanced RNA synthesis occurring before heightened protein synthesis. Inhibiting protein translation blocks miR-122-mediated increases in RNA synthesis, but independently enhances RNA synthesis by releasing ribosomes from viral genomes. Additionally, miR-122 reduces the fraction of viral genomes engaged in protein translation. Depleting AGO2 or PCBP2, which binds HCV RNA in competition with miR-122 and promotes translation, eliminates miR-122 stimulation of RNA synthesis. Thus, by displacing PCBP2, miR-122 reduces HCV genomes engaged in translation while increasing the fraction available for RNA synthesis.

The eponymous member of the interferon regulatory factor (IRF) family, IRF1, was originally identified as a nuclear factor that binds and activates the promoters of type I interferon genes. However, subsequent studies using genetic knockouts or RNAi-mediated depletion of IRF1 provide a much broader view, linking IRF1 to a wide range of functions in protection against invading pathogens. Conserved throughout vertebrate evolution, IRF1 has been shown in recent years to mediate constitutive as well as inducible host defenses against a variety of viruses. Fine-tuning of these ancient IRF1-mediated host defenses, and countering strategies by pathogens to disarm IRF1, play crucial roles in pathogenesis and determining the outcome of infection.