Hepatitis A virus (HAV) is a globally important cause of enterically-transmitted hepatitis. It is one of 9 distinct Hepatovirus species in the Picornaviridae, among which phylogenetic reconstructions suggest multiple past host species jumps. HAV readily infects mice with defective type I interferon responses, suggesting the major barrier preventing human HAV from replicating in a rodent host is an inability to overcome innate immune responses. In prior studies, only a single nonsynonymous mutation of uncertain significance (3D(pol)-R468K) was identified within the genome of wild-type HAV following passage in interferon-receptor knockout mice. Here, we show that R468K and other mutations in the 3D(pol) polymerase (E461D and D473G) are uniformly present in virus recovered from Ifnar1(-/-) mice following intrahepatic injection of HAV RNA. Reverse molecular genetics experiments confirmed RNAs with R468K or D473G mutations were more likely to initiate sustained infection than wild-type RNA in mice. In competition experiments using cell culture-adapted virus, a K468 mutant out-replicated wild-type R468 in murine cells, whereas R468 rapidly replaced K468 in human cells. These 3D(pol) mutations thus promote HAV replication in a host species-specific manner. AlphaFold 3 modeling indicates E461, R468, and D473 are closely positioned on the surface of the 3D(pol) thumb domain, suggesting they modulate interactions with species-specific host factor(s). Proteomics analysis of proteins co-precipitating with HA-3D(pol) expressed in Huh-7.5 cells identified heat shock 70 protein HSPA8 and its co-chaperone, BAG2. HSPA8 is known to be a critical hepatovirus host factor and HAV genome replication is highly dependent upon heat shock chaperone activity. The mouse-adaptive R468K mutation enhances co-immunoprecipitation of 3D(pol) with murine HSPA8 and BAG2, suggesting it facilitates chaperone-dependent acquisition of polymerase function in mouse cells. Our results identify a non-immune barrier to HAV replication in mice and enable future reverse molecular genetics studies in a small animal model.

A common cause of acute hepatitis in humans, hepatitis A virus (HAV) replicates within hepatocytes without inducing cytopathology. Virus is released from infected cells in the absence of cell lysis as quasi-enveloped HAV (eHAV) virions cloaked in host membranes. These virions circulate in blood when exported across the basolateral membrane of hepatocytes but are stripped of their membranes by bile salts when exported across the apical membrane into the biliary system resulting in fecal shedding of abundant naked, nonenveloped virus. This review summarizes the composition and structure of these two distinct types of infectious extracellular hepatovirus virions and outlines the evidence for specific signals within HAV capsid proteins that mediate interactions with the endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated proteins ALIX and HD-PTP play a crucial role in the budding of newly assembled capsids into multivesicular endosomes, the first step in nonlytic release of quasi-enveloped virions from infected cells. This review also considers how eHAV virions enter naïve cells to establish infection in the absence of a virally encoded protein on their surface and compares the role played by quasi-envelopment in the hepatovirus life cycle with the nonlytic release of other types of viruses in extracellular vesicles.

A limited number of studies on human subjects with type-A hepatitis have found a positive correlation between activated natural killer (NK) cell frequencies in blood and the severity of liver injury. However, without knowledge of differences in viral burdens, it remains unclear whether NK cells are elevated due to hepatitis A virus (HAV) replication and ongoing associated injury, or if NK cell responses directly cause pathogenesis. To gain insight into how NK cells respond during the early stages of hepatitis A virus infection, we generated mice that are permissive for HAV infection in the liver but are otherwise immunocompetent. HAV readily infected the liver of these hepatocyte-specific type-I interferon receptor knockout mice (Ifnar1ΔHep), resulting in fecal virus shedding, elevated serum alanine aminotransferase levels, and immune cell infiltrates in the liver. Single-cell RNA sequencing analyses of liver leukocytes from infected Ifnar1ΔHep mice revealed a rapid accumulation of activated NK cells in the liver within 2 days. HAV infection and liver inflammation were brief and substantially reduced in magnitude in these mice compared to global Ifnar1-/- knockout mice that failed to induce substantial antiviral NK cell responses. NK cells were the principal source of interferon (IFN)-γ in Ifnar1ΔHep mice. Ifnar1ΔHep mice that were experimentally depleted of NK cells or IFN-γ showed greatly increased viral replication and liver damage. Moreover, IFN-γ alone was sufficient to suppress HAV replication in primary mouse hepatocytes. Collectively, these findings establish a critical role for type-I IFN induction of NK cells, their expression of IFN-γ, and protection against hepatitis A.IMPORTANCEHepatitis A virus remains a leading cause of foodborne illness among unvaccinated individuals. Infection can result in severe liver injury that can progress to fatal fulminant viral hepatitis. Despite its clinical significance, the molecular, genetic, and cellular factors influencing infection severity remain poorly understood. Previous studies of patient blood have suggested that natural killer (NK) cells cause liver injury during infection. In this study, we used mouse models of infection to characterize early cellular defenses to infection and identified a critical role for NK cells and interferon-γ in conferring rapid immune protection rather than pathogenesis.

Hepatitis E virus (HEV) is a major cause of acute hepatitis and mainly transmitted faecal-orally. HEV particles present in faeces are naked (nHEV), whereas those found in the blood are quasi-enveloped (eHEV) with a cell-derived lipid membrane. Despite its global health impact, the cellular life cycle of HEV remains poorly understood, particularly regarding the mechanisms of viral entry into host cells. To address this knowledge gap, we develop a high content RNA-FISH-based imaging assay that allows for the investigation of the entry pathways of both naked and quasi-enveloped HEV particles. Surprisingly, we find that integrin α3, previously implicated in nHEV cell entry, is not expressed in the cell types that are most permissive for HEV infection. Instead, we identify integrin β1 (ITGB1) pairing with different α-integrins as the key player mediating nHEV cell entry. Our results indicate that the interaction of nHEV with ITGB1 facilitates entry through Rab11-positive recycling endosomes. In contrast, eHEV particles do not interact with ITGB1 and enter cells using a classical endocytic route via Rab5a-positive early endosomes. The entry of both types of HEV particles requires endosomal acidification and proteolytic cleavage by lysosomal cathepsins, which ultimately results in delivery of the HEV genome to the cytoplasm.

HSP90 heat shock chaperones are essential for maintaining cellular proteostasis, as well as the ATP-dependent folding and functional maturation of many viral proteins. As a result, inhibitors of HSP90 have broad antiviral activity, disrupting replication of many viruses at concentrations below those causing cytotoxicity. Among the Picornaviridae, HSP90 inhibitors block replication of multiple Enterovirus, Aphthovirus, and Cardiovirus species, in some cases, by preventing post-translational processing and assembly of P1 capsid proteins. Hepatitis A virus (HAV), classified within the genus Hepatovirus, has been suggested to be an exception among picornaviruses and to replicate independently of HSP90, possibly because its slow translational kinetics could facilitate co-translational folding and assembly of its capsid proteins. However, we show here that HAV replication is highly dependent upon HSP90, both in human hepatocyte-derived cell lines, in which the 50% inhibitory concentration of geldanamycin was 8.7-11.8 nM, and in vivo in Ifnar1-/- mice. Label-free proteomics experiments suggested that HSP90 interacts with capsid proteins or their precursors and may thus facilitate the folding and assembly of capsid proteins, as it does for enteroviruses and aphthoviruses. By contrast, there was no evidence for HSP90 interacting with any nonstructural protein, and HSP90 inhibitors did not impair 3Cpro proteolytic activity. Despite this, and in contrast to previous studies of enteroviruses and aphthoviruses, geldanamycin potently inhibited replication of a subgenomic HAV replicon. We conclude that HAV is no exception from the HSP90-dependent nature of other picornaviruses and indeed is more dependent on HSP90 than other picornaviruses for amplification of its genome.IMPORTANCEHepatitis A virus (HAV), a common cause of acute infectious hepatitis, has been reported to differ from other picornaviruses in not requiring heat shock protein HSP90 for efficient replication. However, we show here that productive HAV infection is highly dependent on HSP90 and that HAV replication is potently blocked both in cell culture and in vivo in the murine liver by chemical inhibitors of HSP90. Such inhibitors also disrupt the replication of a subgenomic HAV RNA replicon, indicating that HSP90 is required for the assembly of functional replication organelles. This highlights a key difference from other picornaviruses for which HSP90 is required primarily, if not exclusively, for the maturation of the P1 capsid proteins.

Both hepatitis B virus (HBV), an hepadnavirus with a DNA genome, and hepatitis A virus (HAV), a picornavirus, require the TRAMP-like host ZCCHC14-TENT4 complex for efficient replication. However, whereas HBV requires the nucleotidyltransferase activity of TENT4 to extend and stabilize the 3′ poly(A) tails of mRNA transcribed from its genome, the role played by TENT4 in HAV replication is uncertain. HAV proteins are synthesized directly from its genomic RNA, which possesses a 3′ poly(A) tail, with its length and composition presumably maintained by 3Dpol-catalyzed RNA transcription during its replicative cycle. Using nanopore long-read sequencing of RNA from infected cells, we confirm here that the length of the HAV 3′ poly(A) tail is not altered by treating infected cells with RG7834, a small molecule TENT4 inhibitor with potent anti-HAV activity. Despite this, TENT4 catalytic activity is essential for HAV replication. Surprisingly, nanopore sequencing revealed a low abundance of HAV subgenomic RNAs (hsRNAs) that extend from the 5′ end of the genome to a site within the 5′ untranslated RNA (5′UTR) immediately downstream of a stem-loop to which the ZCCHC14-TENT4 complex is recruited. These hsRNAs are polyadenylated, and their abundance is sharply reduced by RG7834 treatment, implying they are likely products of TENT4. Similar subgenomic RNAs were not identified in poliovirus-infected cells. hsRNAs are present not only in HAV-infected cell culture but also in the liver of HAV-infected mice, where they represent 1–3% of all HAV transcripts, suggesting their physiological relevance. However, transfecting exogenous hsRNA into TENT4-depleted cells failed to rescue HAV replication, leaving the functional role of hsRNA unresolved. These findings reveal a novel picornaviral subgenomic RNA species while highlighting mechanistic differences in the manner in which HAV and HBV exploit the host ZCCHC4-TENT4 complex for their replication.

Despite the considerable clinical and economic burden imposed by hepatitis A virus (HAV) infection, both globally and in U.S., there are currently no available antiviral therapies for the treatment of type A hepatitis. Here we describe novel third-generation hepato-selective dihydroquinolizinones (HS-DHQs) with cellular uptake mediated by transport via hepatocyte-specific solute organic anion transporter family members 1B1 and 1B3 (OATP1B1-B3). The lead HS-DHQ compound, HS83128, demonstrates robust inhibition of the host cell TENT4A/B terminal nucleotidyltransferases required for efficient HAV RNA synthesis (IC50 6-25nM), and potent antiviral activity against HAV in cell culture (EC50 0.6 nM). Pharmacokinetic studies in CD-1 mice receiving comparable oral doses of HS83128 and a first-generation dihydroquinolizinone, RG7834, revealed a 5-fold increase in intrahepatic drug concentration and more than 10-fold improvement in liver versus nervous system tissue selectivity. Twice-daily oral administration of HS83128 rapidly arrested viral replication in HAV-infected Ifnar1-/- mice, reducing fecal virus shedding and cytokine markers of hepatic inflammation and reversing virus-induced liver injury. The hepato-selective nature of HS83128 may reduce the risk of neurologic and reproductive track toxicities observed with long-term administration of other dihydroquinolizinones, making it a candidate for the first antiviral therapy of hepatitis A.

The overexpression and misfolding of viral proteins in the endoplasmic reticulum (ER) may cause cellular stress, thereby inducing a cytoprotective, proteostatic host response involving phosphorylation of eukaryotic translation initiation factor 2 subunit alpha (eIF2α). Here, we show that hepatitis A virus, a positive-strand RNA virus responsible for infectious hepatitis, adopts a stress-resistant, eIF2α-independent mechanism of translation to ensure the synthesis of viral proteins within the infected liver. Cap-independent translation directed by the hepatovirus internal ribosome entry site and productive hepatovirus infection of mice both require platelet-derived growth factor subunit A (PDGFA)-associated protein 1 (PDAP1), a small phosphoprotein of unknown function with eIF4E-binding activity. PDAP1 also interacts with eIF1A and is essential for translating stress-resistant host messenger RNAs that evade the proteostatic response to ER stress and that encode proteins promoting the survival of stressed cells.

Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis -targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 mu M range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.

ABSTRACT Relatively little is known of the mechanisms underlying hepatitis A virus (HAV) genome replication. Unlike other well-studied picornaviruses, HAV RNA replication requires the zinc finger protein ZCCHC14 and non-canonical TENT4 poly(A) polymerases with which it forms a complex. The ZCCHC14–TENT4 complex binds to a stem-loop located within the internal ribosome entry site (IRES) in the 5’ untranslated RNA (5’UTR) and is essential for viral RNA synthesis, but the underlying mechanism is unknown. Here, we describe how different ZCCHC14 domains contribute to its RNA-binding, TENT4-binding, and HAV host factor activities. We show that the RNA-binding activity of ZCCHC14 requires both a sterile alpha motif (SAM) and a downstream unstructured domain (D4) and that ZCCHC14 contains two TENT4-binding sites: one at the N-terminus and the other around D4. Both RNA-binding and TENT4-binding are required for HAV host factor activity of ZCCHC14. We also demonstrate that the location of the ZCCHC14-binding site within the 5’UTR is critical for its function. Our study provides a novel insight into the function of ZCCHC14 and helps elucidate the mechanism of the ZCCHC14–TENT4 complex in HAV replication. IMPORTANCE The zinc finger protein ZCCHC14 is an essential host factor for both hepatitis A virus (HAV) and hepatitis B virus (HBV). It recruits the non-canonical TENT4 poly(A) polymerases to viral RNAs and most likely also a subset of cellular mRNAs. Little is known about the details of these interactions. We show here the functional domains of ZCCHC14 that are involved in binding to HAV RNA and interactions with TENT4 and describe previously unrecognized peptide sequences that are critical for the HAV host factor activity of ZCCHC14. Our study advances the understanding of the ZCCHC14–TENT4 complex and how it functions in regulating viral and cellular RNAs.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl–coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides. Our data provide insight into how a pathogenic virus alters lipid flux in infected hepatocytes and demonstrate a distinction between lipid species required for viral RNA synthesis versus nonlytic quasi-enveloped virus release.

Background & Aims: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/-mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. Methods: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/-mice.Results: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/-mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/-mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/-mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/-mice.Conclusions: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice.(c) 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Hepatitis C virus (HCV) requires two cellular factors, microRNA-122 (miR-122) and poly(C) binding protein 2 (PCBP2), for optimal replication. These host factors compete for binding to the 5' end of the single-stranded RNA genome to regulate the viral replication cycle. To understand how they interact with the RNA, we measured binding affinities of both factors for an RNA probe representing the 5' 45 nucleotides of the HCV genome (HCV45). Isothermal titration calorimetry revealed two, unequal miR-122 binding sites in HCV45, high-affinity (S1) and low-affinity (S2), differing roughly 100-fold in binding affinity. PCBP2 binds a site overlapping S2 with affinity similar to miR-122 binding to S2. PCBP2 circularizes the genome by also binding to the 3' UTR, bridging the 5' and 3' ends of the genome. By competing with PCBP2 for binding at S2, miR-122 disrupts PCBP2-mediated genome circularization. We show that the viral RNA-dependent RNA polymerase, NS5B, also binds to HCV45, and that the binding affinity of NS5B is increased in the presence of miR-122, suggesting miR-122 promotes recruitment of the polymerase. We propose that competition between miR-122 and PCBP2 for HCV45 functions as a translation-to-replication switch, determining whether the RNA genome templates protein synthesis or RNA replication.

Hepatitis E virus (HEV) is a major cause of acute hepatitis and mainly transmitted faecal-orally. HEV particles in faeces are non-enveloped, while those in the blood possess a cell-derived lipid envelope. Despite being a global health concern, there is limited understanding of the steps in the HEV life cycle, particularly cell entry. A previous study proposed integrin alpha 3 (ITGA3) as a potential host factor for nHEV entry, but the β-integrin partner that co-mediates HEV entry has not been described. To address this knowledge gap and resolve the existing controversies surrounding HEV cell entry, we developed an RNA-FISH-based high-content imaging assay alllowing investigation of the entry pathways of both naked and enveloped HEV particles. Our observations indicate that naked HEV particles interact with the surface receptor integrin beta 1 (ITGB1), which likely facilitates their trafficking through the recycling endosome. In contrast, enveloped HEV particles do not interact with ITGB1 and instead use the classical endocytic pathway via the early endosome. Importantly, both forms of HEV require endosomal acidification and proteolytic cleavage by lysosomal cathepsins, which ultimately results in delivery of the HEV genome to the cytoplasm.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity.

Infectious hepatitis type A and type E are caused by phylogenetically distinct single-stranded, positive-sense RNA viruses that were once considered to be non-enveloped. However, studies show that both are released nonlytically from hepatocytes as 'quasi-enveloped' virions cloaked in host membranes. These virion types predominate in the blood of infected individuals and mediate virus spread within the liver. They lack virally encoded proteins on their surface and are resistant to neutralizing anti-capsid antibodies induced by infection, yet they efficiently enter cells and initiate new rounds of virus replication. In this Review, we discuss the mechanisms by which specific peptide sequences in the capsids of these quasi-enveloped virions mediate their endosomal sorting complexes required for transport (ESCRT)-dependent release from hepatocytes through multivesicular endosomes, what is known about how they enter cells, and the impact of capsid quasi-envelopment on host immunity and pathogenesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5′ untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3′ poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3′ poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC 50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1 −/− mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1(-/-) mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm(3) in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4(+) or CD8(+) T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifn gamma, Tnf alpha, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1(-/-) mice and that macro- phages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1(-/-) mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1(-/-) mice and may have relevance to relapsing hepatitis A in humans.